Prior to the present invention, numerous reports have appeared in the literature which cited the reaction of lipopolysaccharide (LPS) with cyclic anhydrides, especially phthalic and succinic anhydride. However, with the exception of one reference, no mention has been made of combining monophosphoryl lipid A (MPL) with these or other anhydrides.
In an article by A. Hasegawa, et al, J. Carbohydrate Chem.5: 371, there is disclosed the covalent coupling of muramyl dipeptide (MDP) to a derivative of lipid A corresponding to the non-reducing glucosamine residue. This lipid A derivative, designated GLA-27 in the reference, was blocked at both the phosphate and at all other potentially reactive positions except for the hydroxyl to which coupling was desired (i.e. the primary hydroxyl at C6). The coupling strategy employed in this reference involved introducing a free carboxyl group into GLA-27 at C6 via succinic anhydride and a free amine into MDP, and then forming an amide linkage between these two groups. A key difference between the teachings of this reference and those of the present invention are that in the reference, the lipid A derivative had to be completely blocked in order to avoid side reactions during the condensation (i.e., amide bond forming) step. The present invention avoids this difficulty by introducing a free amino group into MPL. Coupling of MPL to other components is then achieved using reagents which react specifically with amines (e.g. aldehydes), or else activating an appropriate group on the other component prior to combining with the amino-MPL.
Acylation of lipopolysaccharides by cyclic anhydrides probably occurs in the O-antigen and core regions which are absent in monophosphoryl lipid A. The purpose of acylating LPS in these prior art references was to attenuate its toxicity, not to introduce a functional group for the purpose of forming covalent conjugates with other materials.
Accordingly, one or more of the following objects will be achieved by the practice of the present invention. It is an object of the invention to provide certain novel derivatives of monophosphoryl lipid A and a process for their preparation. Another object of this invention is to provide a novel process for the preparation of derivatives of monophosphoryl lipid A which does not require the blocking of any functional groups in order to introduce the desired groups into the molecule.
A still further object of the invention is to provide novel derivatives which are conjugates of monophosphoryl lipid A with biologically active materials such as antigens, antibodies, immunomodulators and the like. Another object is to provide novel conjugates of the derivatives with biological compounds such as antigens, which exhibit enhanced biological activity.
A further object of this invention is to provide a process for the preparation of derivatives of monophosphoryl lipid A in relatively pure form and substantially free of undesirable reaction by-products.
Another object of the invention is to provide methods for using the conjugates. These and other objects will readily become apparent to those skilled in the art in light of the teachings contained herein.